Yanhua Hu

Activation of PDGF receptor α in vascular smooth muscle cells by mechanical stress

Hypertension increases mechanical force on the arterial wall by as much as 30%, resulting in marked alterations in signal transductions and gene expression in vascular smooth muscle cells (VSMCs) that contribute to matrix protein synthesis, cell proliferation, and differentiation. How the mechanical stimuli are converted into a biological signal in cells has yet to be studied. We investigated the role of both cyclic strain and shear stresses in initiating the cellular signaling on cultured VSMCs and found that mechanical forces evoked activation of mitogen‐activated protein kinases, followed by enhanced DNA binding activity of transcription factor AP‐1. Physical forces rapidly induced phosphorylation of platelet‐derived growth factor receptor (PDGFR) α, an activated state. When GRB2, an adapter protein, was immunoprecipitated from treated VSMCs followed by Western blot analysis with anti‐phosphotyrosine, ‐PDGFRα, and ‐GRB2 antibodies, respectively, phosphotyrosine positive staining was observed on PDGFRα bands of the same blot in stretch‐stressed VSMCs, supporting the mechanical stress‐induced activation of PDGFRα. Conditioned medium from stretch‐stressed VSMCs did not result in PDGFRα phosphorylation, and antibodies binding to all forms of PDGFs did not block stress‐induced PDGFRα activation. Thus, mechanical stresses may directly perturb the cell surface or alter receptor conformation, thereby initiating signaling pathways normally used by growth factors.—Hu, Y., Böck, Güunther, Wick, G., Xu, Q., Activation of PDGF receptor α in vascular smooth muscle cells by mechanical stress. FASEB J. 12, 1135–1142 (1998)

Mouse Model of Venous Bypass Graft Arteriosclerosis

Saphenous vein grafts are widely used for treatment of severe atherosclerosis via aortocoronary bypass surgery, a procedure often complicated by later occlusion of the graft vessel. Because the molecular mechanisms of this process remain largely unknown, quantitative models of venous bypass graft arteriosclerosis in transgenic mice could be useful to study this process at the genetic level. We describe herein a new model of vein grafts in the mouse that allows us to take advantage of transgenic, knockout, or mutant animals. Autologous or isogeneic vessels of the external jugular or vena cava veins were end-to-end grafted into carotid arteries of C57BL/6J mice. Vessel wall thickening was observed as early as 1 week after surgery and progressed to 4-, 10-, 15-, and 18-fold original thickness in grafted veins at age 2, 4, 8, and 16 weeks, respectively. The lumen of grafted veins was significantly narrowed because of neointima hyperplasia. Histological and immunohistochemical analyses revealed three lesion processes: marked loss of smooth muscle cells in vein segments 1 and 2 weeks after grafting, massive infiltration of mononuclear cells (CD11b/18+) in the vessel wall between 2 and 4 weeks, and a significant proliferation of vascular smooth muscle cells (alpha-actin+) to constitute neointimal lesions between 4 and 16 weeks. Similar vein graft lesions were obtained when external jugular veins or vena cava were isografted into carotid arteries of C57BL/6J mice. Moreover, no significant intima hyperplasia in vein-to-vein isografts was found, although there was leukocyte infiltration in the vessel wall. Thus, this model, which reproduces many of the features of human vein graft arteriosclerosis, should prove useful for our understanding of the mechanism of vein graft disease and to evaluate the effects of drugs and gene therapy on vascular diseases.

Biomechanical stress-induced apoptosis in vein grafts involves p38 mitogen-activated protein kinases

The present study was designed to investigate whether apoptosis occurs in early‐stage vein grafts and to determine the mechanisms by which mechanical stress contributes to apoptosis in vascular smooth muscle cells (SMCs). Apoptosis in vessel walls of mouse vein grafts was confirmed by morphological changes and by terminal deoxynucleotidyl transferase‐mediated dUTP‐biotin nick end labeling (TUNEL). TUNEL+ cells in vein grafts 1, 4, and 8 wk postoperatively was 13%, 29%, and 21%, respectively, and apoptosis occurred mainly in veins grafted to arteries, remaining unchanged in vein‐to‐vein grafts. When mouse, rat, and human arterial SMCs were cultured on a flexible membrane and subjected to cyclic strain stress, apoptosis was observed in a time‐ and strength‐dependent manner. All three types of SMCs showed apoptotic death as confirmed by TUNEL, propidium iodide, and annexin V staining. To further study the signal pathways leading to apoptosis, activities of p38, a subfamily of mitogen‐activated protein kinases (MAPKs), were determined. Mechanical stress resulted in p38 MAPK activation, reaching high levels within 8 min. SB 202190, a specific inhibitor for p38 MAPKs, prevented SMC apoptosis in response to mechanical stress. SMC lines stably transfected with a dominant negative rac, an upstream signal transducer, or overexpressing MAPK phosphatase‐1, a negative regulator for MAPKs, completely inhibited mechanical stress stimulated p38 activation and abolished mechanical stress‐induced apoptosis. Thus, we provide solid evidence that one of the earliest events in venous bypass grafts is apoptosis, in which mechanical stress‐induced p38‐MAPK activation is responsible for transducing signals leading to apoptosis.—Mayr, M., Li, C., Zou, Y., Huemer, U., Hu, Y., Xu, Q. Biomechanical stress‐induced apoptosis in vein grafts involves p38 mitogen‐activated protein kinases. FASEB J. 15, 261–270 (2000)

Nitric oxide induces heat-shock protein 70 expression in vascular smooth muscle cells via activation of heat shock factor 1.

Current data suggest that nitric oxide (NO) is a double-edged sword that could result in relaxation and/or cytotoxicity of vascular smooth muscle cells (SMCs) via cGMP- dependent or -independent signal pathways. Stress or heat shock proteins (hsps) have been shown to be augmented in arterial SMCs during acute hypertension and atherosclerosis, both conditions that are believed to correlate with disturbed NO production. In the present study, we demonstrate that NO generated from sodium nitroprusside (SNP), S-nitroso-N-acetylpenicillamine, and spermine/nitric oxide complex leads to hsp70 induction in cultured SMCs. Western blot analysis demonstrated that hsp70 protein expression peaked between 6 and 12 h after treatment with SNP, and elevated protein levels were preceded by induction of hsp70 mRNA within 3 h. Induction of hsp70 mRNA was associated with the activation of heat shock transcription factor 1 (HSF1), suggesting that the response was regulated at the transcriptional level. HSF1 activation was completely blocked by hemoglobin, dithiothreitol, and cycloheximide, suggesting that the protein damage and nascent polypeptide formation induced by NO may initiate this activation. Furthermore, SMCs pretreated with heat shock (42 degrees C) for 30 min were significantly protected from death induced by NO. Thus, we provide evidence that NO induces hsp70 expression in SMCs via HSF1 activation. Induction of hsp70 could be important in protecting SMCs from injury resulting from NO stimulation.

Increased Expression and Activation of Stress-Activated Protein Kinases/c-Jun NH2-Terminal Protein Kinases in Atherosclerotic Lesions Coincide with p53

Hyperlipidemia alters gene expression of arterial endothelial and smooth muscle cells (SMCs) and induces atherosclerotic lesions, in which cell proliferation and apoptosis co-exist. The signal transduction pathways that mediate these responses in the vessel wall in vivo have yet to be identified. Stress-activated protein kinases (SAPKs) or c-Jun NH(2)-terminal protein kinases (JNKs) are thought to be crucial in transmitting transmembrane signals required for cell differentiation and apoptosis in vitro. In the present study, we investigated the localization and activity of SAPK/JNK in atherosclerotic lesions of cholesterol-fed rabbits. Immunofluorescence analysis revealed abundant and heterogeneous distribution of pan-SAPK/JNK and phosphorylated SAPK/JNK, which were mainly localized in cell nuclei of the lesional cap and basal regions. Double staining of the lesions demonstrated that a portion of alpha-actin(+) SMCs and RAM11(+) macrophages contained abundant phosphorylated SAPK/JNK proteins. SAPK/JNK protein levels in protein extracts from atherosclerotic lesions were two- to threefold higher than the vessels of chow-fed rabbits. SAPK/JNK activities were elevated three- to fivefold higher than the normal vessels. Interestingly, increased SAPK/JNK in lesions was co-localized or coincided with high levels of transcription factor p53 as identified by double labeling and immunoprecipitation. Abundant pro-apoptotic protein BAX and BCL-X(S) were also observed. Furthermore, low-density lipoprotein (LDL) and oxidized LDL stimulated SAPK/JNK activation in cultured SMCs in a time- and dose-dependent manner. LDL also induced SAPK/JNK activation in vascular SMCs derived from LDL-receptor-deficient Watanabe rabbits, indicating a LDL-receptor-independent process. Thus, SAPK/JNK persistently hyperexpressed and activated in lesions may play a key role in mediating cell differentiation and apoptosis during the development of atherosclerosis via activation of transcription factor p53.

Rapid Development of Vein Graft Atheroma in ApoE-Deficient Mice

Several animal models manifesting lesions resembling neointimal hyperplasia of human vein grafts have been developed, but no spontaneous atheromatous lesions in their vein grafts have been observed. We developed and here characterize a new animal model of vein graft atheroma, a maturated atherosclerotic plaque, in apoE-deficient mice. The lesion displayed classical complex morphological features and heterogeneous cellular compositions and consisted of a fibrous cap, infiltrated mononuclear cells, foam cells, cholesterol crystal structure, necrotic core with calcification, and neovasculature. Cell component analysis revealed smooth muscle cells (SMCs) localized in the cap region, macrophages which made up a large portion of the lesions, and CD4+ T cells scattered under the cap. Importantly, apoptotic/necrotic cells determined by TUNEL assay in vein grafts into apoE−/− mice were significantly higher than wild-type mice, although a similar number of proliferating cell nuclear antigen-positive cells in both types of lesions was found. Interestingly, vascular SMCs cultivated from aortas of apoE-deficient mice showed a high rate of spontaneous apoptosis/necrosis and a higher rate of cell death stimulated by a nitric oxide donor, sodium nitroprusside, H 2 O 2 , and oxidized low density lipoprotein (LDL), although no difference in proliferation of both SMCs incubated with platelet-derived growth factor, angiotensin II, LDL, and oxidized LDL was seen. Thus, the pathogenic mechanisms of vein graft atheroma involve increased intimal cell death initiated by biomechanical stress and amplified by hypercholesterolemia, which leads to continuous recruitment of blood mononuclear cells to constitute atheromatous lesions. This mouse model resembling human vein graft disease has many advantages over other animal models.

Disturbed Immuno-Endocrine Communication via the Hypothalamo-Pituitary-Adrenal Axis in Autoimmune Disease

Previous studies in our laboratory demonstrated an altered immuno-endocrine feedback communication via the hypothalamo-pituitary-adrenal (HPA) axis, which may be an important modulatory factor in the development of spontaneous autoimmune thyroiditis in Obese strain (OS) chickens. These birds show a significantly lower, or even absent, increase in serum glucocorticoid levels in response to an intravenous injection of antigen or conditioned medium (CM) from mitogen-stimulated spleen cells known to contain glucocorticoid-increasing factors (GIFs), notably interleukin-1 (IL-1). The present study was aimed at investigating this feedback regulation in animal models with spontaneous systemic autoimmune diseases, such as the UCD-200 chicken, which serves as a model for human scleroderma, and various murine lupus models. In contrast to OS chickens, UCD-200 chickens displayed a nearly normal plasma corticosterone surge in response to CM, and IL-1 was again identified as the primary GIF in CM. Recombinant IL-1 also induced a drastic increase in plasma corticosterone levels in various strains of normal mice. A similar increase was observed in the bacterial lipopolysaccharide-resistant C3H/HeJ strain, thus excluding the possibility of bacterial endotoxin contamination. However, in young lupus-prone (NZB/W)F1 and MRL/MP-lpr mice, a significantly lower increase in plasma corticosterone levels was observed after injection of recombinant IL-1, suggesting a deficient immuno-endocrine communication via the HPA loop in this instance as well. Detailed studies to identify further cytokines with GIF activity in the avian and murine systems showed that both IL-6 and tumor necrosis factor-alpha could induce increased plasma corticosterone levels in mice, but not in chickens. IL-3, IL-8, transforming growth factor-beta, interferon-gamma and granulocyte-macrophage colony-stimulating factor were devoid of GIF activity in both chickens and mice.

Disturbed Immunoendocrine Communication via the Hypothalamo–Pituitary–Adrenal Axis in Murine Lupus

Immune reactions and mitogen stimulation of mammals and chickens lead to an increase of glucocorticoid (GC) plasma levels concomitant with the immune response. Interleukin (IL) 1, one of the most important glucocorticoid increasing factors produced by cells of the immune system, acts via the hypothalamo-pituitary-adrenal (HPA) axis. This pattern of immunoendocrine feedback communication is altered in autoimmune disease (AID) and represents a possible site of action for GC therapy. In the present study we investigated the role and possible underlying mechanisms of a disturbed immunoendocrine communication via the HPA axis in murine lupus. We analyzed the response to recombinant human (rhu) IL-1alpha in AID-prone mice [NZB, NZW, (NZB/NZW)F1, MRL/MP-lpr] in comparison to nonautoimmune, normal control mice (Swiss, C3H/HeJ, MRL/MP-+/+) at different levels of the HPA axis. To this end, we quantified the plasma levels of ACTH, corticosterone, and corticosterone-binding globulin (CBG) and determined various pathology parameters for autoimmunity. AID-prone mice produced nearly the same levels of plasma corticosterone after injection of rhu IL-1alpha as normal mice, but had baseline corticosterone levels consistently higher, thus resulting in significantly lower corticosterone increasing ratios. ACTH levels increased after rhu IL-1alpha injection, but there was no clearcut difference in the increasing ratios of AID-prone and normal strains. CBG levels showed no difference. As expected, there was a correlation of pathology parameters for autoimmunity and the altered immunomodulatory response to rhu IL-1alpha per group. On an individual basis, there was no such correlation. In conclusion, our results confirm the existence of a disturbed immunoendocrine communication in AID-prone mice. This disturbance clearly differs from individual to individual and also among different types of AID.

Interleukin-1 receptor deficiency in brains from NZB and (NZB/NZW) F1 autoimmune mice

Interleukin-1 receptors (IL-1R) are expressed in the brain and the anterior pituitary of normal mice (C3H/He, Swiss), and appear to be involved in the neuroendocrine control of the immune response. Here we have studied the IL-1R density in the brain and the pituitary from several strains of autoimmune mice (NZB, (NZB/NZW)F1, MRL/MP-lpr), using quantitative autoradiography with recombinant human [125I]IL-1 alpha as a ligand. IL-1R was similar in the brain of C3H/He, Swiss and NZW (controls) and MRL/MP-lpr mice. In NZB mice a profound deficit (10% of control mice) in IL-1R was observed exclusively in the dentate gyrus. In (NZB/NZW)F1 the deficit was about 50%. These observations were independent of sex and age. Pituitary receptors were not affected in all the strains except NZW (30% increase). Competition experiments demonstrated that the affinity of IL-1R was not modified in dentate gyrus of (NZB/NZW)F1 and NZW mice. Thus, the number of IL-1R was the only parameter affected. This deficit was not reversed by corticosterone treatment (0.2 mg/20 g body weight, i.p.) and was poorly modified by lipopolysaccharide treatment (0.1 mg/20 g body weight, i.p.) compared to C3H/He mice. In conclusion, this central IL-1R deficit is unlikely to be the consequence of occupancy by abnormal synthesis of brain IL-1. This abnormality is tissue-specific with hereditary autosomal transmission. The role of central IL-1R in neuroimmunoendocrine interactions and in autoimmunity remains to be clarified.

The role of the immunoendocrine interaction via the hypothalamo-pituitary-adrenal axis in autoimmune disease Emphasis on the obese strain chicken model.

The key to the molecular basis of the pathogenesis of autoimmune disease (AID) certainly lies in the identification of genes coding for the altered immune response and those responsible for the susceptibility of the target organ to the autoimmune attack. The elucidation of nonessential modulatory factors is, however, also of great importance, because it is via these routes that an effective AID therapy is practical at the present time. Hormones in general, and those affecting the hypothalamo-pituitary-adrenal (HPA) axis in particular, are among the best candidates for more rational new therapeutic approaches. Studies of immune-endocrine communication in animal models with spontaneously occurring organ-specific or systemic autoimmune diseases are ideal for this kind of investigation, since immunologic studies commencing prior to onset of the AID in question can be performed.